Many inorganics are human carcinogens and pose major hazards to after environmental or occupational exposure. Arsenic and cadmium are two of the most important inorganic carcinogens and with arsenic alone over 100 million people world-wide are exposed to clearly unhealthy levels via contaminated drinking water in what is likely the worst mass poisoning in human history. Defining carcinogenic mechanisms will greatly aid in designing prevention or intervention strategies and in assigning appropriate levels of risk to these exposures. The primary goal of the ICS is to define the molecular mechanisms of carcinogenic action of arsenic and cadmium under the project titled Molecular Mechanisms of Inorganic Carcinogenesis. Since they can impact carcinogenicity and potentially provide a means for prevention or intervention, mechanisms of adaptation and acquired tolerance are studied as well. Emphasis is placed on key factors that dictate sensitivity, such as early-life exposure and poor expression of critical adaptive genes. These inorganics attack various targets sites in humans. Arsenic causes skin, urinary bladder, lung, kidney, liver, and prostatic malignancies and likely uterine cancers. Cadmium is primarily associated with human lung, prostatic and kidney cancers and recently pancreatic cancers. All these sites may have distinct differences in mode of action and adaption. Thus, various in vitro and in vivo model systems have been developed to study important molecular targets in targets tissues, with an emphasis on human relevance. These inorganic carcinogens likely have multiple mechanisms that are site and cell specific. A reproducible rodent model where inorganic arsenic acts a complete carcinogen has been developed in which brief in utero arsenic exposure in mice leads to tumors or proliferative lesions of the urogenital system, liver, lung and adrenal in the offspring as adults. The urogenital system lesions include transplacental arsenic-induced or initiated tumors of the ovaries, uterus, vagina, and bladder and proliferative lesions of the kidney. These results are in accord with human studies that indicate the liver, urinary bladder, lung, kidney and uterus are target tissues of arsenic carcinogenesis. Molecular mechanism studies indicate disruption of estrogen signaling by in utero exposure to arsenic contributes to the liver, lung and urogenital system malignancies, in part through aberrant activation of estrogen receptor-a. Indeed, we find that tumors and proliferative lesions of the urogenital system, including the uterus, ovary, vagina and urinary bladder, are greatly enhanced by postnatal exposure to synthetic estrogens like diethylstilbestrol. We also find evidence of aberrant estrogen signaling in arsenic exposed human liver. Further molecular characterization of arsenic-induced in utero tumor initiation is underway, including aberrant gene imprinting, using this model of arsenic carcinogenesis. We hypothesize that arsenic in utero may attack a critical pool of stem cells in target organs and induces aberrant genetic reprograming as part of its carcinogenic mechanism. These studies have important public health implications, including the identification of early life period as a time of very high sensitivity to arsenic. Human data is now emerging that fetal and/or early life exposure to arsenic is clearly carcinogenic. Further study will include prenatal arsenic exposure combined with exposures to urinary bladder and renal tumor promoters in mice to enhance the carcinogenic response to arsenic in these key human target organs. Various in vitro cell transformation model systems have also been developed to study inorganic carcinogenesis. In doing these studies we select cells with relevance to the human targets of arsenic, cadmium or lead carcinogenesis, and use low-level exposures for long periods, which approximates typical human exposures and avoids supra-physiological responses associated with acute high doses that could have limited relevance to the carcinogenic process. A human prostate epithelial cell line has been malignantly transformed with cadmium and arsenic, both potential human prostatic carcinogens. Additional work indicates the arsenic and cadmium transformants both acquire androgen independence, an event associated with a very poor clinical prognosis in prostatic cancer patients, largely through androgen receptor by-pass related mechanisms. Molecular dissection of the events associated with arsenic- or cadmium-induced malignant transformation in this and other human cell lines will continue with a focus of aberrant expression of genes critical to the carcinogenic process. In addition, a human prostate stem cell line has been developed and we have show it is malignantly transformed by arsenic through a combination of selection due to a survival advantage over mature heterogenous populations, and due to aberrant stimulation of self-replication genes typical for stem cells populations. This creates the distinct possibility that arsenic causes cancer by attack on stem cell populations. Indeed, in a mouse in vivo model of skin cancer, we have found that arsenic in utero causes over production of cancer stem cells in adult skin squamous cell carcinoma apparently by distorting fetal skin stem cell dynamics. Other tumors model systems are now being investogated. Furthermore, we have successfully transformed a human pancreatic ductal cell with cadmium, which fortifies a possible role of cadmium in this deadly disease. In this case it appears that cadmium initially kills back stem cells. However, after this bottle-neck effect the stem cell emerge transformed and capable of malignant behavior. This is consistent with cadmium as a single dose carcinogen. We are now seeing this effect in other lines with cadmium. Many cell biomethylate arsenic using specific methyltransferases S-adenosylmethionine (SAM) as the methyl group donor. We have found that cells that do not biomethylate arsenic cannot induce oxidative DNA damage (ODD). This is a very important finding because many target cells do not biomethylate arsenic but none-the-less become malignantly transformed by the metalloid. This likely indicates multiple mechanisms are operative with arsenic, some of which are genotoxic (ODD) and some of which may not be.